Hair cells are the primary sensory receptors of the inner ear, and are responsible for detecting sound as well as balance. A number of factors are attributable to hearing loss;this includes drugs that elicit ototoxicity through mechanisms that are largely unclear. Our lab is investigating the causative role of these drugs in hair cell death in hopes of identifying protective strategies. Because it is challenging to study ototoxin-induced hair cell death in mammals, we employ the zebrafish system as a tool to study such events. In addition to hair cells of the inner ear, zebrafish also possess hair cells on their surface in a sensory system termed the lateral line. While their external location enable zebrafish to sense environmental cues, we as researchers can easily observe and image intracellular events with little perturbation. Because lateral line hair cells resemble hair cells of the mammalian inner ear both structurally and in their response to ototoxins, zebrafish are ideal for the study of signaling and cell death pathways that affect hair cells. We have begun to examine such pathways in earnest, and have started to focus on the role of Ca2+ signaling in hair cell death. Despite its well-known role in many intracellular pathways, including pro-apoptotic ones, Ca2+ has not been extensively examined in hair cells following exposure to ototoxic agents. I propose to examine the role of intracellular Ca2+ in hair cell death utilizing the many benefits of the zebrafish lateral line system. I have taken advantage of previous dose-response characterizations performed in our lab to treat zebrafish with doses of ototoxin that kill approximately 50% of lateral line hair cells, and quantify differences in Ca2+ levels between hair cells that die and those that survive. My initial analysis has identified that intracellular Ca2+ levels are significantly higher in dying hair cells exposed to either the aminoglycoside neomycin or the anticancer therapeutic cisplatin. The work proposed here aims to reveal subcellular events that lead to this increase, as well as to better understand why a seemingly uniform population of cells exhibits a differential cell death response. I will utilize genetically encoded Ca2+ indicators targeted to various subcellular compartments within the hair cell to visualize these events in real-time. Much of this work is expected to define additional steps within the pathway of hair cell death. These studies should provide new insight into methods of ototoxin action as well as the potential to block hair cell death during ototoxin exposure. Pharmacological agents exist that are capable of regulating the subcellular flow of Ca2+ within the cell, and understanding how these ototoxins affect intracellular Ca2+ holds promise for future therapies designed to protect human hair cells from death. PUBLIC HEALTH RELEVANCE: Death of hair cells within the inner ear is the prominent cause of hearing loss within the population. One such causative factor stems from the side effects of common therapeutic agents, which include aminoglycoside antibiotics and platinum-based anticancer therapeutics. Because of their cost and/or effectiveness, these drugs continue to be used despite their ototoxic effects. Thus, the side effects of these drugs remain a significant health issue. There is therefore a need to better understand the mechanisms underlying ototoxin-induced hair cell death. This proposal is aimed at monitoring the response of hair cell death pathways to ototoxins in real- time, and using these findings to identify ways to protect hair cells during treatment with therapeutic agents that possess ototoxic side effects.